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WO2018180568A1 - 2-désoxy-scyllo-inosose synthase de type mutant - Google Patents

2-désoxy-scyllo-inosose synthase de type mutant Download PDF

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WO2018180568A1
WO2018180568A1 PCT/JP2018/010349 JP2018010349W WO2018180568A1 WO 2018180568 A1 WO2018180568 A1 WO 2018180568A1 JP 2018010349 W JP2018010349 W JP 2018010349W WO 2018180568 A1 WO2018180568 A1 WO 2018180568A1
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amino acid
doi
glucose
transformant
gene
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Japanese (ja)
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洋暁 高久
晴丈 山崎
光史 和田
大輔 宮澤
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NIIGATA INSTITUTE OF SCIENCE AND TECHNOLOGY
Mitsui Chemicals Inc
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NIIGATA INSTITUTE OF SCIENCE AND TECHNOLOGY
Mitsui Chemicals Inc
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Priority to EP18777395.7A priority Critical patent/EP3604526A4/fr
Priority to US16/497,970 priority patent/US11499175B2/en
Priority to CN201880020313.XA priority patent/CN110462041B/zh
Priority to JP2019509266A priority patent/JP6989746B2/ja
Priority to KR1020197029474A priority patent/KR102336516B1/ko
Publication of WO2018180568A1 publication Critical patent/WO2018180568A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
    • C12Y402/03124—2-Deoxy-scyllo-inosose synthase (4.2.3.124)

Definitions

  • the present disclosure includes, for example, a modified 2-deoxy-siro-inosose (hereinafter referred to as “DOI”) synthase, a gene encoding the modified DOI synthase, an expression cassette including the gene, a vector including the expression cassette,
  • DOI 2-deoxy-siro-inosose
  • the present invention relates to a transformant containing the vector, a method for producing a modified DOI synthase using the transformant, and a method for producing DOI.
  • DOI a chiral compound having a carbon 6-membered ring skeleton
  • DOI can be synthetically converted into dihydric phenols such as catechol and droquinone, and hydroxyhydroquinone.
  • catechol and droquinone dihydric phenols
  • hydroxyhydroquinone dihydric phenols
  • Kakinuma et al., Tetrahedron Letters, 2000, vol.41, p.1935 discloses synthesizing and converting DOI to catechol.
  • Catechol is used as a raw material for neurological drugs, raw materials for food fragrances, antioxidants for hair care products, etc.
  • hydroquinone is used for raw materials such as hemostatics and analgesics, and cosmetics such as whitening agents. It is a high substance.
  • DOI can also be converted into carbaglucose, which is a pseudo sugar, and is a versatile intermediate material.
  • Japanese Patent Application Laid-Open No. 2005-053899 discloses synthesizing carbaglucose using DOI as a raw material.
  • DOI synthase gene (glucose-6-phosphate as DOI) from microorganisms belonging to Bacillus circulans. It describes that a large amount of recombinant DOI synthase was obtained by cloning btrC) encoding an enzyme that catalyzes the conversion reaction, and expressing and purifying the gene in E. coli. Further, Japanese Patent Application Laid-Open No.
  • 2014-066453 discloses a two-stage enzyme reaction in which hexokinase or polyphosphate glucokinase and DOI synthase are allowed to act on glucose, or a one-stage enzyme in which DOI synthase is allowed to act on glucose-6-phosphate. It discloses that DOI can be synthesized by reaction.
  • Hirayama et al., J. Antibiot., 2005, vol.58, p.766 disclose DOI synthase derived from Streptomyces fradiae, Subba et al., Mol.Cells, 2005, vol.
  • DOI synthase derived from Streptomyces ribosidiphycus and Khale et al., Arch.Biochem.Biophys., 2004, vol.429, p.204 are Streptomyces kanamyceticus.
  • Unwin et al., J. Antibiot., 2004, vol.57, p.436 disclose DOI synthase derived from micromonospora and echinospora, and Kharel et al., FEMS Microbiol. Lett., 2004, vol.230, p.185 disclose DOI synthase derived from Streptomyces teneblarius, and Hirayama et al., J. Antibiot., 2006, vol.59, p.358 Disclosed is DOI synthase derived from Aroteicus hindustanus.
  • JP2013-135597A discloses a thermostable DOI synthetase having a specific amino acid sequence. Tamigai et al., Biosci. Biotechnol. Biochem., 2010, vol.74, p.1215 are Bacillus Circus. It describes the role of the BtrC2 protein associated with Lance's DOI synthase.
  • WO 2006/109479 and Kogure et al., J. Biotechnol., 2007, vol.129, p.502 disclose an expression cassette consisting of a gene for DOI synthase, which is an engineering of intracellular sugar metabolism. Attempts have also been made to increase production by modification.
  • WO 2010/053052 has a gene encoding at least sucrose hydrolase (CscA) among the sucrose non-PTS genes, and a 2-deoxy-siro-inosose (DOI) production system is provided or enhanced.
  • CscA sucrose hydrolase
  • DOI 2-deoxy-siro-inosose
  • DOI-producing E. coli further having a system for enhancing sugar uptake ability is disclosed.
  • the present inventors succeeded in modifying DOI synthase into an enzyme having higher DOI synthesizing activity by using an evolutionary engineering technique. Moreover, it succeeded in manufacturing DOI more efficiently than before using the transformant containing the gene which codes the modified enzyme obtained by this method.
  • An expression cassette comprising the polynucleotide according to (4), a promoter sequence linked upstream of the polynucleotide, and a terminator sequence linked downstream of the polynucleotide.
  • a transformant transformed with the vector according to (6) A method for producing a polypeptide having an enzymatic activity for producing 2-deoxy-siro-inosose from glucose-6-phosphate, comprising culturing the transformant according to (7).
  • Mutant DOI synthetase gene clone in the process from preparation of mutant DOI synthase gene clone library using error-prone PCR to isolation of DOI high production DOI synthase gene mutant clone (1, 2 and 3) It is a figure which shows preparation of a library. It is a figure which shows the primary selection in the process from preparation of the mutant
  • coli GI724 ⁇ pgi ⁇ zwf ⁇ pgm strain ( ⁇ ) containing pGADP-btrC ( ⁇ ) and E. coli GI724 ⁇ pgi ⁇ zwf ⁇ pgm strain ( ⁇ ) containing one of pGADP-mbtrC (later known as pGADP-btrC (W293R)) (2 ⁇ YT + 2% manure , 30 ° C.) is a diagram showing a time course of the DOI production amount in the culture medium. It is a figure which shows DOI synthetase activity of (left) wild type DOI synthetase (WT) and (right) mutant type DOI synthetase (W293R). E.
  • coli GI724 ⁇ pgi ⁇ zwf ⁇ pgm strain ( ⁇ ) containing pGADP-mbtrC (later known as pGADP-btrC (W293R / Y37F)), pGADP-mbtrC (later known as ⁇ GAipGg7 including ⁇ GAipgg7 including ⁇ gg7, including pGADP-btrC (W293R / A290T) + ), E.
  • coli GI7 containing pGADP-mbtrC (later identified as pGADP-btrC (W293R / H319R)) 4 ⁇ pgi ⁇ zwf ⁇ pgm strain ( ⁇ ) of the culture is a diagram showing the time course of the DOI production in the medium due to (2 ⁇ YT + 2% glucose + 2% mannitol, 50ml, 30 °C).
  • the DOI production rate can be improved by high DOI synthesis activity, and glucose-6-phosphate can be efficiently converted into DOI in a short time.
  • a modified synthase having high DOI synthesis activity can be produced by expressing a modified DOI synthase gene in a host cell.
  • DOI can be manufactured from glucose with high efficiency using this modified DOI synthetase gene.
  • DOI can be synthetically converted into dihydric phenols such as catechol and hydroquinone and hydroxyhydroquinone (see FIG. 1). According to one embodiment of the present disclosure, it is possible to easily and efficiently produce DOI, which is expected to be widely used as a raw material for producing pharmaceuticals and industrial products, simply and in large quantities. For example, 1,2,4-trihydroxybenzene, which is expected to be widely used as a raw material for producing pharmaceuticals and industrial products, can be produced from the DOI thus produced.
  • the modified DOI synthase according to the present disclosure is a polypeptide having at least one amino acid mutation of the following (a) to (e) in the following amino acid sequence (A1) or (A2).
  • A1 Amino acid sequence of SEQ ID NO: 1
  • A2) Amino acid sequence of a polypeptide having an enzymatic activity to produce 2-deoxy-siro-inosose from glucose-6-phosphate, and the amino acid sequence of SEQ ID NO: 1
  • amino acid sequence having 80% or more sequence identity (a) amino acid mutation in which the amino acid residue corresponding to the 14th asparagine residue from the N-terminal in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine
  • b) An amino acid mutation in which the amino acid residue corresponding to the 37th tyrosine residue from the N-terminal in the amino acid sequence of SEQ ID NO: 1 is substituted with phenylalanine
  • c) 290th alanine
  • No acid mutation An amino acid mutation in which the amino acid residue corresponding to the 293th tryptophan residue from the N-terminal in the amino acid sequence of SEQ ID NO: 1 is substituted with arginine (e) N-terminal in the amino acid sequence of SEQ ID NO: 1 Amino acid mutation in which an amino acid residue corresponding to the 319th histidine residue from arginine is substituted with arginine in the alignment.
  • a modified DOI synthase having a specific amino acid residue substitution is improved by the amino acid residue substitution.
  • Has DOI synthesis activity has DOI synthesis activity.
  • the modified DOI synthase according to the present disclosure is referred to as a polypeptide having an amino acid sequence in which at least one amino acid mutation is introduced from the following (a) to (e) with respect to the amino acid sequence of (A1) or (A2):
  • “at least one amino acid mutation in the following (a) to (e) is introduced into the amino acid sequence of (A1) or (A2)” means the final amino acid sequence. It is used only for identification, and does not limit the amino acid sequence serving as a starting point and the actual sequence modification process.
  • the amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of DOI synthase derived from Bacillus circulans, and is encoded by the btrC gene.
  • the modified DOI synthase has an amino acid sequence different from the amino acid sequence of SEQ ID NO: 1, and is also referred to as a mutant DOI synthase in the present disclosure.
  • the amino acid sequence of SEQ ID NO: 1 is shown in Table 1 below.
  • Enzyme activity to produce 2-deoxy-siro-inosose from glucose-6-phosphate is 50 mM Phosphate Buffer (pH 7.7), 5 mM glucose-6-phosphate, 5 mM ⁇ -NAD + , 0.2 mM CoCl 2.
  • a solution consisting of 6H 2 O and 10 ⁇ g of DOI synthase to be measured is reacted at 46 ° C. for 5 minutes. After the reaction, the reaction solution is treated with phenol / chloroform to remove proteins, and the aqueous layer fraction after centrifugation is 10 ⁇ l. Can be measured by quantifying DOI by HPLC and calculating activity.
  • sequence identity of amino acid sequences can be evaluated with default parameters using, for example, the BLAST (registered trademark, National Library of Medicine) program.
  • the amino acid residue corresponding to the amino acid residue at a specific position in the amino acid sequence of SEQ ID NO: 1 is, for example, BLAST (registered trademark, National Library of Medicine)
  • BLAST registered trademark, National Library of Medicine
  • the amino acid sequence of SEQ ID NO: 1 was aligned with the amino acid sequence of (A1) or (A2) by the program (default parameter), it was found to correspond to the amino acid residue at a specific position in SEQ ID NO: 1. It refers to an amino acid residue in the amino acid sequence of (A1) or (A2).
  • the sequence identity with the amino acid sequence of SEQ ID NO: 1 contained in the amino acid sequence (A2) may be 85% or more, 90% or more, or 95% or more.
  • the amino acid sequence of (A2) may be an amino acid sequence having a sequence modification within a range that does not lose DOI synthase activity in the amino acid sequence of SEQ ID NO: 1. That is, it may be an amino acid sequence obtained by modifying the amino acid sequence of SEQ ID NO: 1 within a range that does not lose DOI synthase activity. Such modifications include insertion, deletion, substitution of amino acid residues and addition of additional amino acid residues at the N-terminal or C-terminal or both of the amino acid sequence.
  • each of the insertions, deletions and substitutions is, for example, 1 to 30 amino acid residues, or 1 to 20 amino acid residues.
  • 1 to 10 amino acid residues, alternatively 1 to 5 amino acid residues, and the total number of insertions, deletions and substitutions of amino acid residues is, for example, 1 to 50 amino acid residues, or 1 to 30 amino acid residues Alternatively, it may be 1 to 10 amino acid residues, alternatively 1 to 5 amino acid residues.
  • the number of amino acid residues added to the terminal is, for example, 1 to 50 amino acid residues, alternatively 1 to 30 amino acid residues, alternatively 1 to 10 amino acid residues, or 1 to 5 per terminal if present. It may be an amino acid residue. Such additional amino acid residues may form a signal sequence for extracellular secretion or the like. Examples of signal sequences include E. coli OmpA signal sequences.
  • All of the amino acid mutations (a) to (e) are amino acid mutations that increase DOI synthase activity.
  • the modified DOI synthetase according to the present disclosure may have only one of the amino acid mutations (a) to (e) or may have two or more.
  • the modified DOI synthase may have two of the amino acid mutations (a) to (e), may have three, or may have four. You may have five.
  • the modified DOI synthase has at least one of the amino acid mutations of (d) and (e), and further comprises at least one of the amino acid mutations of (a) to (c). You may have.
  • the modified DOI synthase has the amino acid mutation of (d), and further has at least one of the amino acid mutations of (a), (b), (c) and (e). In yet another embodiment, the modified DOI synthase has an amino acid mutation of (d) and further has at least one of the amino acid mutations of (a), (b), and (e). In this case, the amino acid mutation (c) may be further included. In yet another embodiment, the modified DOI synthase has an amino acid mutation of (d) and further has at least one of the amino acid mutations of (a) and (e). In this case, it may further have at least one of the amino acid mutations (b) and (c).
  • the modified DOI synthase has an amino acid mutation of (d) and an amino acid mutation of (e). In this case, it may further have at least one of the amino acid mutations (a) to (c). Further, a polypeptide having 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 and having an enzyme activity for producing 2-deoxy-siro-inosose from glucose-6-phosphate is related to the function of the enzyme.
  • the amino acid mutations (a) to (e) described above have the effect of increasing the DOI synthase activity even for such a polypeptide. Play.
  • a modified DOI synthetase having improved DOI synthetase activity can be obtained by having one or more of the amino acid mutations (a) to (e).
  • the modified DOI synthetase preferably has higher DOI synthesizing activity than the DOI synthetase having the amino acid sequence of SEQ ID NO: 1 (also referred to as wild-type DOI synthetase in the present disclosure).
  • the modified DOI synthetase preferably has a DOI synthesizing activity that is 1.1 times or more higher than that of the DOI synthetase having the amino acid sequence of SEQ ID NO: 1, more preferably a DOI synthesizing activity that is 1.2 times or more higher.
  • DOI synthetic activity More preferably 1.3 times higher DOI synthetic activity, more preferably 1.4 times higher DOI synthetic activity, more preferably 1.5 times higher DOI synthetic activity, Preferably, it has a DOI synthesis activity 1.6 times higher, more preferably 1.7 times higher DOI synthesis activity, more preferably 1.8 times higher DOI synthesis activity.
  • amino acid mutations (a) to (e) above were obtained by evolution engineering modification of DOI synthase.
  • Evolutionary engineering modification means that a gene that encodes a target protein is artificially mutagenized in vitro to select a protein that has been modified to a desired property, and then the target protein molecule is modified. Refers to technology.
  • the introduction of random mutations into the target enzyme gene can be achieved by treating the microorganism carrying the target enzyme gene with an alkylating reagent (N-methyl-N'-nitro-N-nitrosoguanidine, etc.), oxidative nucleobase It can be performed by treatment with a deamination reagent (nitrous acid, etc.), irradiation with radiation (ultraviolet rays, X-rays, etc.), or random mutagenesis using PCR.
  • an alkylating reagent N-methyl-N'-nitro-N-nitrosoguanidine, etc.
  • oxidative nucleobase oxidative nucleobase
  • a deamination reagent nitrogen acid, etc.
  • irradiation with radiation ultraviolet rays, X-rays, etc.
  • random mutagenesis using PCR.
  • a DNA fragment containing the target enzyme gene is used as a template, and in the amplification process of the gene, a PCR reaction is performed under conditions that reduce the accuracy of DNA replication by DNA polymerase. This can be done by performing error-prone PCR that accumulates errors in the sequence. In error-prone PCR, the accuracy of DNA polymerase is reduced and mutations are introduced by adding manganese ions to the reaction solution or breaking the balance of the concentrations of the four types of deoxyribonucleic acid (dNTP). be able to.
  • dNTP deoxyribonucleic acid
  • a plasmid containing the gene for example, pLEX-btrC described in WO 2006/109479
  • DNA PCR may be carried out under conditions that reduce the accuracy of DNA polymerase using the gene amplification primer with the fragment as a template. Examples of conditions for reducing the accuracy of the DNA polymerase include conditions described in Example 1 described later.
  • the modified enzyme gene group obtained by random mutagenesis can be screened using as an index whether or not it has an improved function with respect to a desired property. For example, by measuring the DOI synthetic activity of the modified enzyme group expressed by the modified enzyme gene group, the modified enzyme having improved DOI synthetic activity than before the introduction of mutation can be selected. In this way, a modified enzyme with improved activity for the desired properties can be obtained. By further introducing random mutations into the gene encoding the obtained modified enzyme and screening in the same manner as described above, a modified enzyme having further improved activity with respect to the desired properties can be obtained.
  • Evolutionary engineering modification makes it possible to obtain a modified enzyme having improved properties without knowing the position of the active center in the enzyme.
  • such an improvement can be achieved cumulatively by amino acid mutations or combinations thereof at amino acid residue positions whose relationship with function is not known in advance. From this, the modification
  • the gene encoding the modified DOI synthetase according to the present disclosure may be any nucleic acid encoding the modified DOI synthetase.
  • the nucleotide sequence of a nucleic acid encoding a particular amino acid sequence can be varied within the codon degeneracy. In this case, it is preferable from the viewpoint of gene expression efficiency to use a codon that is frequently used in the microorganism that is the host of the recombinant microorganism.
  • a polynucleotide having a base sequence encoding the amino acid sequence of the modified DOI synthase is provided.
  • the nucleotide sequence of the gene can also be designed based on the codon table from the amino acid sequence to be encoded.
  • the designed nucleotide sequence may be obtained by modifying a known nucleotide sequence using a gene recombination technique, or may be obtained by chemically synthesizing the nucleotide sequence. Examples of the method for modifying the nucleotide sequence include site-directed mutagenesis (Kramer, W. and Frita, HJ, Methods in Enzymology, vol. 154, P.
  • a host / vector system a system such as bacteria or yeast can be used, but is not particularly limited as long as a gene having a mutation can be efficiently expressed and produced.
  • the obtained mutated PCR fragment is ligated to an expression vector having a promoter and terminator necessary for expression and capable of being expressed in the host, and introduced into the host.
  • the gene expression cassette according to the present disclosure is not particularly limited as long as the gene encoding the above-mentioned modified DOI synthase can be expressed in the host cell described below.
  • the gene expression cassette may contain, for example, one or more of a promoter, an enhancer, RBS (ribosome binding sequence), a terminator and the like in addition to the nucleic acid sequence encoding the modified DOI synthase.
  • the gene expression cassette preferably contains a promoter upstream of the nucleic acid sequence and a terminator downstream of the nucleic acid sequence in addition to the nucleic acid sequence encoding the modified DOI synthase.
  • a DNA sequence such as a promoter, an enhancer, and an RBS (ribosome binding site) upstream of the DNA sequence encoding the modified DOI synthase
  • a terminator DNA sequence may be linked downstream (3 ′ end side) of the DNA sequence encoding the modified DOI synthase.
  • Each of these elements is not particularly limited as long as it is a sequence that exhibits a desired function in E. coli.
  • a promoter capable of inducing expression with an inducer such as IPTG (isopropylthiogalactopyranoside) may be used.
  • examples of the promoter include lactose operon promoter, tryptophan operon promoter, previous two fusion promoters, lambda phage promoter, glyceraldehyde-3-phosphate dehydrogenase gene promoter, glutamate
  • examples include a decarboxylase gene promoter, a gadA promoter, and an alcohol dehydrogenase (ADH1) promoter.
  • the terminator is not particularly limited, and for example, an rrn terminator, an AspA terminator, or the like can be used.
  • examples of the ribosome binding site include AGGAG having a Shine-Dalgarno (SD) sequence.
  • a known enhancer can be used as the enhancer.
  • the modified DOI synthetase gene expression vector according to the present disclosure is not particularly limited as long as it has a gene encoding the above-mentioned modified DOI synthetase and can be expressed in the host cell described below.
  • the modified DOI synthetase gene expression vector preferably comprises the modified DOI synthetase gene expression cassette.
  • E. coli when E. coli is used, various expression vectors have been constructed for efficient gene expression. Lactose operon promoter, tryptophan operon promoter, previous two fusion promoters, ⁇ phage promoter, glyceraldehyde-3-phosphate dehydrogenase gene promoter, glutamate decarboxylase gene promoter, gadA promoter, alcohol dehydrogenase (ADH1) promoter, etc.
  • a modified DOI synthase expression vector can be constructed in which a mutation-treated gene is connected downstream of the gene and a terminator is connected downstream of the gene. As the terminator, an rrn terminator, an AspA terminator, or the like can be used, but is not particularly limited.
  • a vector constructed for gene recombination from a phage or plasmid capable of autonomously growing in a host cell is suitable.
  • the phage include Lambda gt10 and Lambda gt11 when Escherichia coli is used as a host cell.
  • plasmids for example, when Eschencia coli is used as a host cell, pBTrp2, pBTac1, pBTac2 (Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen-1), pGEM-1 Promega), pQE-8, pQE-30 (QIAGEN), pBluescript II SK (+), pBluescript II SK (-) (Stratagene), pET-3 (Novagen), pUC18, pSTV28, pSTV, pSTV28 Examples include pUC118 (Takara Shuzo), pLEX (Invitrogen), pQE80L (QIAGEN), pBR322, and the like.
  • the modified DOI synthetase gene expression vector may have a promoter for transcription of DNA encoding the modified DOI synthetase.
  • the promoter include the promoters described above.
  • the modified DOI synthase gene expression vector may contain a ribosome binding sequence.
  • ribosome binding sequences include Shine-Dalgarno sequences, and it is preferable to use a plasmid in which the SD sequence and the start codon are adjusted to an appropriate distance (eg, 6 to 18 nucleotides).
  • the N-terminus of the target protein may be fused to the N-terminal portion of another protein encoded by the expression vector.
  • a terminator is not necessarily required for expression of the target protein, but it is desirable to place a terminator directly under the structural gene.
  • a vector DNA fragment can be obtained by cleaving the above-mentioned vector with the restriction enzyme used for excision of the inserted DNA, but it is not necessarily the same as the restriction enzyme used for excision of the inserted DNA. It is not necessary to use the restriction enzyme.
  • the method for binding the inserted DNA fragment and the vector DNA fragment may be a method using a known DNA ligase. For example, after annealing of the sticky end of the inserted DNA fragment and the sticky end of the vector DNA fragment, Recombinant DNA of a DNA fragment to be inserted and a vector DNA fragment is prepared by using an appropriate DNA ligase. If necessary, after annealing, it can be transferred to a host cell such as a microorganism and recombinant DNA can be produced using in vivo DNA ligase.
  • a transformant expressing the modified DOI synthase gene can be obtained by introducing the expression vector thus prepared into a host capable of replicating and maintaining. Subsequently, the modification
  • the transformant according to the present disclosure is a transformant including the modified DOI synthase gene expression vector according to the present disclosure.
  • the host cell used for the preparation of the transformant may be any host cell as long as the recombinant DNA can be stably and autonomously propagated and a foreign DNA trait can be expressed.
  • the host cell is preferably a microbial cell.
  • the microorganism cell may be a eukaryotic cell (for example, yeast) or a prokaryotic cell. Examples of host cells include Escherichia coli cells, but are not particularly limited to E.
  • coli cells cells of Escherichia bacteria, cells of Bacillus bacteria such as Bacillus subtilis, Pseudomonas Bacteria cells such as genus bacteria, yeast cells such as Saccharomyces genus, Pichia genus, Candida genus, filamentous fungi cells such as Aspergillus can be used.
  • the host cell is preferably a host cell that accumulates a large amount of glucose-6-phosphate serving as a substrate for DOI synthase.
  • host cells include strains in which the pgi gene encoding glucose phosphate isomerase is disrupted in E. coli (for example, the E. coli GI724 ⁇ pgi strain described in WO 2006/109479), the pgi gene and glucose- A strain in which the zwf gene encoding 6-phosphate dehydrogenase is disrupted (for example, E.
  • a disrupted strain see FIG. 2 (for example, E. coli GI724 ⁇ pgi ⁇ zwf ⁇ pgm strain described in WO 2006/109479).
  • the host cell is preferably a cell having a gene encoding an enzyme that produces glucose-6-phosphate from glucose, such as glk. Further, the host cell may be further given the ability to take extracellular glucose into the cell as it is, and for this purpose, for example, a glucose transport promoting protein gene may be further introduced. Examples of the glucose transport promoting protein gene include glf derived from Zymomonas mobilis. In addition, the host cell may be further provided with improved utilization of fructose and sucrose. For this purpose, for example, a sucrose hydrolase gene may be further introduced. Examples of the sucrose hydrolase gene include cscA derived from E. coli O-157. An example of a vector having such a gene is the plasmid vector pGAP-btrC-cscA-glf described in International Publication No. 2010/053052.
  • a competent cell method using calcium treatment, an electroporation method, or the like can be used.
  • competent cells E. coli DH5 ⁇ competent cells and the like can be used.
  • the culture conditions for the transformant are the same as those for the original host microorganism, and known conditions can be used.
  • various carbon sources, nitrogen sources, inorganic salts and organic nutrient sources can be arbitrarily used.
  • carbon sources that can be used include glucose, sucrose, molasses, and fats.
  • nitrogen source include ammonium salts such as ammonia, ammonium chloride, ammonium sulfate, and ammonium phosphate, peptone, meat extract, yeast extract, and the like.
  • inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride.
  • examples of other organic nutrient sources include amino acids such as glycine, alanine, serine, threonine, and proline, and vitamins such as vitamin B1, vitamin B12, and vitamin C.
  • the medium contains appropriate amounts of carbon source, nitrogen source, inorganic substance and other nutrients
  • a synthetic medium or a natural medium can be used.
  • the medium include LB liquid medium, RM liquid medium, 2 ⁇ YT liquid medium, L agar medium, RMM liquid medium, and the like.
  • an LB agar plate may be used.
  • a medium containing the corresponding drug is used, and the selection marker is nutrient-free. If required, use a medium that does not contain the corresponding nutrients.
  • the culture conditions may be appropriately selected depending on the type of culture medium and the culture method, and are not particularly limited as long as the transformant grows.
  • the culture temperature may be any temperature at which the transformant can grow.
  • the pH at the time of culture may be a pH at which the transformant can grow.
  • the culture time is not particularly limited as long as it can efficiently produce DOI synthase.
  • the culture temperature may be, for example, a temperature in the range of 20 ° C. to 45 ° C., a temperature in the range of 25 ° C. to 35 ° C., or a temperature in the range of 24 ° C. to 37 ° C. Good.
  • the pH of the medium may be selected, for example, in the range of 4 to 8, may be in the range of 5 to 8, and may be in the range of 6.5 to 8.
  • the culture may be performed aerobically or anaerobically depending on the type of microorganism.
  • the culture period may be, for example, 1 hour to 7 days, 6 hours to 60 hours, or 12 hours to 30 hours.
  • the culture period may be set so that the production amount of the modified DOI synthase is maximized.
  • the time required for the cultivation is It may be within 48 hours.
  • the culture time may be in the range of 0.5 hours to 30 hours. Culturing can be performed in a liquid medium containing the above-described culture components by using a normal culture method such as shaking culture, aeration-agitation culture, continuous culture, or fed-batch culture.
  • the obtained transformant is cultured under conditions where DOI can be produced, and the production state of DOI in the culture solution is examined.
  • the production status of DOI can be examined with a gas chromatograph mass spectrometer or a high performance liquid chromatograph analyzer.
  • Such production amount analysis can be performed with reference to Kogure et al., J. Biotechnol., 2007, vol.129, p.502.
  • an expression vector possessed by the transformant is extracted, and a base sequence of a gene encoding the enzyme is determined.
  • the predicted amino acid sequence of the enzyme can then be compared with the amino acid sequence of the wild-type enzyme to identify which amino acids contribute to the modification of the enzyme properties.
  • site-directed mutagenesis is performed to determine how much each amino acid substitution contributes to the modification of the properties of the enzyme. Any one amino acid can be replaced with another amino acid by the method and examined.
  • Enzymological properties of the mutant enzymes obtained above are as follows: after separation and purification of each mutant enzyme, the specific activity, substrate specificity, optimum temperature, optimum pH, etc. are examined and compared with the wild-type enzyme. This makes it possible to verify changes in the properties of the enzyme.
  • the modified DOI synthase produced by the transformant can be used by directly collecting a culture solution containing the transformant in the culture.
  • a transformant can also be extract
  • the collected transformant is disrupted by a mechanical method or an enzymatic method such as lysozyme, and if necessary, a chelating agent such as Ethylenediaminetic acid (EDTA) and / or a surfactant is added to solubilize the polypeptide, It can be separated and collected as a solution.
  • EDTA Ethylenediaminetic acid
  • the method for producing a polypeptide having DOI synthesis activity according to the present disclosure includes culturing the transformant according to the present disclosure.
  • the medium used for culturing the transformant, the culture conditions, the culture method, and the like are as described in the description of the transformant according to the present disclosure.
  • the culture conditions are not particularly limited as long as the host cells can grow and produce a protein having DOI synthesis activity.
  • the gene encoding the modified DOI synthase on the vector according to the present disclosure is expressed, and the modified DOI synthase is generated.
  • the modified DOI synthase can be obtained by culturing within 48 hours at a pH in the range of 6 to 8 and a temperature in the range of 25 to 40 ° C. in an appropriate medium under aerobic conditions.
  • the produced modified DOI synthase is contained in at least one of the transformant cells (for example, Escherichia coli cells) and the culture medium.
  • the cells and medium of the transformant may be used directly in the DOI synthesis reaction without purification, or the modified DOI synthase may be purified from the medium.
  • the modified DOI synthase in the cell can also be recovered by disrupting or dissolving the cell.
  • the cells may be separated from the medium by centrifugation or the like, and the separated cells may be used for the DOI synthesis reaction, or the cells may be dried, frozen or lyophilized and stored.
  • the separated cells may be disrupted or lysed, and the released DOI synthase may be used as it is for the DOI synthesis reaction, or the released DOI synthase may be purified.
  • general purification methods such as centrifugation, salting-out, desalting, chromatography, electrophoresis, ultrafiltration, etc. can be used under appropriately controlled conditions.
  • DOI synthase can be purified by lysis of cells using Lysis buffer, fixation of DOI synthase to Ni-NTA agarose, and elution with elution buffer.
  • the DOI production method includes a modified DOI synthase according to the present disclosure, a transformant according to the present disclosure, a culture of the transformant, or a transformant or a processed product of the culture.
  • a method for producing DOI comprising converting glucose or glucose-6-phosphate to DOI by contacting with 6-phosphate.
  • the culture of the transformant refers to a product obtained by culturing the transformant and comprising cells and the surrounding medium.
  • the culture may not be used.
  • dried or frozen transformant cells prepared in advance may be added directly to the reaction system.
  • the medium, culture conditions, culture method, and the like for obtaining a transformant culture are the same as the culture medium, culture conditions, culture method, and the like that can be used for culturing the transformant.
  • the culture conditions are not particularly limited as long as the host cells can grow and produce a protein having DOI synthesis activity.
  • the treated product of the transformant refers to a product obtained by subjecting the transformant to any treatment within a range not losing the activity of the modified DOI synthase produced by the transformant.
  • treatment include heat treatment, cooling treatment, mechanical destruction, ultrasonic treatment, freeze-thaw treatment, drying treatment, pressurization or decompression treatment, osmotic pressure treatment, self-digestion, surfactant treatment and enzyme treatment.
  • the treated product of the culture refers to a product obtained by subjecting the cultured product of the transformant to any treatment within a range not losing the activity of the modified DOI synthase produced by the transformant.
  • treatment include heat treatment, cooling treatment, cell mechanical destruction, ultrasonic treatment, freeze-thaw treatment, drying treatment, pressurization or decompression treatment, osmotic pressure treatment, cell autolysis, surfactant treatment,
  • the treatment include one or more selected from the group consisting of enzyme treatment (for example, cell destruction treatment), cell separation treatment, purification treatment, and extraction treatment.
  • the transformant cells may be separated from the medium or the like, and the separated cells may be added to the reaction system.
  • separation means such as filtration or centrifugation can be used.
  • separating a modified DOI synthetase from a contaminant may be performed, and the solution containing the enzyme obtained by this refinement
  • an extract obtained by extracting the culture with an organic solvent such as methanol or acetonitrile or a mixed solvent of an organic solvent and water may be added to the reaction system.
  • Such a purified product or extract may be free from transformant cells. Even if the cells of the transformant do not exist, it can be used for the reaction as long as the enzyme activity remains.
  • the above-mentioned cell disruption or lysis treatment can be performed by disrupting the cell membrane of the transformant according to a known method such as lysozyme treatment, freeze-thawing, or ultrasonic disruption.
  • the contact of the transformant according to the present disclosure, the culture of the transformant or the treated product of the transformant or the culture with glucose or glucose-6-phosphate is performed under the following conditions. Is preferred.
  • the contact is preferably performed in a solution containing glucose or glucose-6-phosphate as a substrate.
  • the solution may contain both glucose and glucose-6-phosphate.
  • the reaction is preferably performed in the presence of a coenzyme such as NAD or NADP in view of reaction efficiency.
  • Reaction conditions are not particularly limited as long as the above reaction proceeds.
  • the pH of the solution is not particularly limited as long as the enzyme activity of the modified DOI synthase is maintained.
  • the pH during the reaction is preferably in the range of 4.0 to 9.0.
  • the pH is preferably in the range of 5.0 to 8.0, and more preferably in the range of 6.0 to 8.0.
  • the temperature of the solution is not particularly limited as long as the enzyme activity of the modified DOI synthase is maintained, but is preferably a temperature in the range of 10 ° C to 50 ° C, more preferably a temperature in the range of 20 ° C to 45 ° C. Even more preferred is a temperature within the range of 30 ° C to 42 ° C.
  • the solution medium water or an aqueous medium, an organic solvent, or a mixed solution of water or an aqueous medium and an organic solvent is used.
  • aqueous medium for example, a buffer solution such as a phosphate buffer solution, a HEPES (N-2-hydroxyethylpiperazine-N-ethanesulfonic acid) buffer solution, or a tris [tris (hydroxymethyl) aminomethane] hydrochloric acid buffer solution is used.
  • Any organic solvent may be used as long as it does not inhibit the reaction. For example, acetone, ethyl acetate, dimethyl sulfoxide, xylene, methanol, ethanol, butanol and the like are used.
  • the solution may be a liquid medium.
  • the contact of the transformant according to the present disclosure, the culture of the transformant or the treated product of the transformant or the culture with glucose or glucose-6-phosphate is performed under shaking or stirring.
  • such contact can be done in solution.
  • glucose or glucose-6-phosphate in the form of a substrate solution or in the form of a solid It may be added.
  • An acid or an alkali may be added at the start of the reaction or during the reaction in order to maintain the pH of the reaction solution within an appropriate range.
  • alkalis examples include alkali metal hydroxides such as lithium hydroxide, sodium hydroxide, and potassium hydroxide, as well as ammonium hydroxide, calcium hydroxide, dipotassium phosphate, and disodium phosphate. , Potassium pyrophosphate, ammonia and the like which are dissolved in water to make the liquid basic.
  • acids examples include hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid and the like.
  • the contact may be performed, for example, in an air atmosphere or a deoxygenated atmosphere.
  • the deoxygenated atmosphere can be achieved by substitution with an inert gas, reduced pressure, boiling, or a combination thereof. It is preferable to use at least substitution with an inert gas, that is, an inert gas atmosphere.
  • the inert gas include nitrogen gas, helium gas, argon gas, carbon dioxide gas, and the like, preferably nitrogen gas.
  • the transformant, the transformant culture, or the transformant or the treated product of the transformant used contains the modified DOI synthase.
  • the modified DOI synthetase present in the reaction solution acts to produce DOI with high production efficiency.
  • a substance involved in the reaction is continuously supplied by metabolism. From the standpoint of being able to do, it is preferable to include the transformant in a living state.
  • the transformant, the culture of the transformant or the treated product of the transformant or the culture may be added all at once at the start of the reaction, or divided or continuously during the reaction. You may add.
  • glucose or glucose-6-phosphate as a raw material may be added all at once at the start of the reaction, or may be added dividedly or continuously during the reaction.
  • the concentration of glucose or glucose-6-phosphate in the reaction solution is, for example, 0.1% by mass to 20% by mass, alternatively 0.5% by mass to 15% by mass, or alternatively 2% by mass to 10% by mass. It may be.
  • Examples of the method of bringing the transformant, the transformant culture, or the transformant or the treated product of the transformant into contact with glucose or glucose-6-phosphate include the transformant, the transformation A culture of the body or the transformant or a treated product of the culture is added to a solution containing glucose or glucose-6-phosphate, and the reaction is allowed to proceed with stirring, the culture of the transformant or the trait A method of allowing a transformant or a treated product of the culture to be added to a solution containing glucose or glucose-6-phosphate and allowing the reaction to proceed while shaking, the transformant, the culture of the transformant, or the transformation Examples thereof include a method in which a body or a processed product of the culture and pyridoxine or a salt thereof are sufficiently mixed in a solution and then allowed to stand to allow the reaction to proceed.
  • the transformant, the culture of the transformant, or the transformant or a processed product of the culture is added to a solution containing glucose or glucose-6-phosphate and stirred. And a method of allowing the reaction to proceed.
  • reaction vessel that can be used for the reaction.
  • the transformant added, the transformant culture, or the transformant or the treated product of the transformant and the solution containing pyridoxine or a salt thereof can be stirred and mixed so that they are sufficiently mixed.
  • the reaction vessel has a temperature control function so that it can be kept within the optimum temperature range of DOI synthase.
  • the contact time (reaction time) between the transformant, the transformant culture, or the transformant or the treated product of the transformant and glucose or glucose-6-phosphate is the enzyme of the modified DOI synthase.
  • reaction time There is no particular limitation as long as the activity is maintained, but it may be, for example, 30 minutes to 100 hours, or 2 hours to 50 hours.
  • the reaction may be performed in a batch mode, or in the course of the reaction, it may be performed in a semi-batch mode in which one or both of the substrate and the microorganism, the culture or the treated product are sequentially added, or performed in a continuous mode. Also good.
  • the upper limit of the reaction time is not particularly limited. Not.
  • glucose or glucose-6-phosphate may be added continuously.
  • the method for producing DOI includes culturing a transformant according to the present disclosure in a medium.
  • the medium is preferably a liquid medium and preferably contains glucose or glucose-6-phosphate.
  • the medium may further contain fructose.
  • the medium, culture conditions, culture method, and the like that can be used the above-described culture medium, culture conditions, culture method, and the like that can be used for culturing the transformant can be applied.
  • a 2 ⁇ YT liquid medium can be used.
  • Glucose or glucose-6-phosphate may be added to such a medium so that a desired concentration of glucose or glucose-6-phosphate is included in the medium.
  • the culture temperature may be, for example, a temperature within the range of 25 ° C. to 35 ° C., and the culture time may be, for example, 5 hours to 30 hours.
  • the concentration of glucose or glucose-6-phosphate in the medium is, for example, 0.1% by mass to 20% by mass, alternatively 0.5% by mass to 15% by mass, alternatively 1.5% by mass to 10% by mass. %.
  • Such culture also produces DOI due to the presence of the transformant.
  • pre-culture Prior to culture for DOI production (hereinafter also referred to as main culture), pre-culture may be performed on the transformant.
  • the medium used for the pre-culture may be a medium different from the medium used for the culture for DOI production.
  • the difference in the medium may be a difference in the basic medium or a difference in the concentration of glucose or glucose-6-phosphate.
  • the medium, culture conditions, culture method, and the like used for the preculture the above-described medium, culture conditions, culture method, and the like that can be used for culturing the transformant can be applied.
  • the medium used for the pre-culture may be a medium containing neither glucose nor glucose-6-phosphate.
  • Examples of the medium used for the preculture include an RM liquid medium and an RMM liquid medium, but may be a 2 ⁇ YT liquid medium.
  • the preculture may be performed until the state of the transformant is stabilized, but the preculture time is, for example, 3 hours to 48 hours, or may be 8 hours to 30 hours.
  • the transformant after the preculture may be added as it is to the medium for main culture as it is, separated from the medium used for the preculture by centrifugation or the like, and then added to the medium for main culture. Also good.
  • the amount of the culture solution in the main culture can be larger than the amount of the culture solution in the preculture, for example, 10 times or more by volume, or 20 times or more.
  • the main culture may be performed in a batch mode, or may be performed in a semi-batch mode in which glucose or glucose-6-phosphate is sequentially added during the reaction, or glucose or glucose-6-phosphate is continuously added. You may carry out by a continuous type. In the case of a semi-batch type or a continuous type, an operation such as supplying a new raw material is performed, and thus the upper limit of the reaction time is not particularly limited.
  • the use of the transformant according to the present disclosure, the culture, or the processed product of the transformant or the culture using glucose or glucose-6-phosphate as a raw material enables high production efficiency of DOI.
  • the DOI obtained by the above method can be converted into catechol and used as a raw material for neurological drugs, raw materials for food fragrances, antioxidants for hair care products, etc., and converted into hydroquinone to stop hemostatics, analgesics, etc. It can be used as a raw material for cosmetics and cosmetics such as whitening agents.
  • TAB 1,2,4-trihydroxybenzene
  • TGB 1,2,4-triglycidyloxybenzene
  • TGB is excellent in heat resistance and is a low-viscosity liquid at room temperature, and can be used over a wide range of materials such as sealing materials for electronic components, circuit substrates, adhesives, coating materials, paints, and matrix resins for composite materials.
  • process is not limited to an independent process, and even if it cannot be clearly distinguished from other processes, the term “process” is used if the intended purpose of the process is achieved. included.
  • a numerical range indicated by using “to” indicates a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
  • the amount of each component in the composition when there are a plurality of substances corresponding to each component in the composition, the plurality of the components present in the composition unless otherwise specified. It means the total amount of substance.
  • Example 1 ⁇ Construction of mutant DOI synthase gene clone library by error-prone PCR> 3A to 3D show the steps from the preparation of a mutant DOI synthase gene clone library using error-prone PCR to the isolation (1, 2, and 3 selection) of DOI high-production DOI synthase gene mutant clones. It was.
  • a mutation was randomly introduced into the gene (btrC) encoding the amino acid sequence of DOI synthase derived from Bacillus circulans shown in SEQ ID NO: 1 by error-prone PCR.
  • Primer 1 (5′-acgcgtcgacatgacgactaaacaaatttg-3 ′) corresponding to the nucleotide sequence obtained by adding the SalI restriction enzyme site upstream of the start codon of the btrC gene using the NdeI-XbaI site of the site as a template
  • primer 2 (5′-aaaactgcagttacagcccttccgga-3
  • PCR is held at 94 ° C for 2 minutes and 30 seconds, followed by 30 cycles of heat denaturation at 94 ° C for 20 seconds, annealing at 50 ° C for 25 seconds, and DNA extension reaction at 72 ° C for 1 minute and 10 seconds. The mixture was further kept at 72 ° C. for 3 minutes, and the resulting PCR amplification product was kept at 4 ° C.
  • the DNA fragment thus PCR amplified was treated with phenol / chloroform, centrifuged, and the supernatant was ethanol precipitated to recover the DNA fragment.
  • the recovered DNA fragment was digested with restriction enzymes SalI and PstI, and purified and isolated by agarose gel electrophoresis to obtain a DNA fragment of a mutant DOI synthase gene.
  • an expression vector for expressing the DNA fragment of the mutant DOI synthase gene in a host cell was constructed.
  • pLEX vector Invitrogen
  • primer 3 5′-atggtaccgagctcggatcc-3 ′
  • primer 4 shown in SEQ ID NO: 5 corresponding to the nucleotide sequence added with an XbaI restriction enzyme site on the 5 ′ side
  • PCR amplification was performed using 5′-ctagtctagactaggagataatttatcaccgcag-3 ′.
  • KOD polymerase TOYOBO
  • PCR reaction conditions were maintained at 94 ° C. for 2 minutes, followed by 30 cycles of heat denaturation at 94 ° C. for 30 seconds, annealing at 52 ° C. for 30 seconds, and DNA extension at 68 ° C. for 1 minute. Further, it was kept at 68 ° C. for 2 minutes, and the obtained PCR amplification product was kept at 4 ° C.
  • the DNA fragment thus amplified by PCR was treated with phenol / chloroform, centrifuged, and the supernatant was ethanol precipitated to recover the DNA fragment.
  • the recovered DNA fragment was digested with restriction enzymes SalI and XbaI and purified and isolated by agarose gel electrophoresis to obtain an expression vector DNA fragment.
  • a gadA promoter was obtained as a promoter for expressing a DNA fragment of the mutant DOI synthase gene in a host cell (E. coli). Specifically, the gadA promoter was obtained as follows. Using a chromosomal DNA of E.
  • a primer 5 (5′-ctagtctagagtcgtttttctgct-3 ′) shown in SEQ ID NO: 6 corresponding to a nucleotide sequence added with an XbaI restriction enzyme site on the 5 ′ side and a SalI restriction enzyme site on the 5 ′ side PCR amplification was performed using the primer 6 (5′-acgcgtcgacttcgaactccttaaatttattttgaaggc-3 ′) shown in SEQ ID NO: 7 corresponding to the added nucleotide sequence.
  • KOD polymerase (TOYOBO) was used for PCR amplification.
  • PCR reaction conditions were maintained at 94 ° C for 2 minutes, followed by 30 cycles of heat denaturation at 94 ° C for 30 seconds, annealing at 50 ° C for 30 seconds, and DNA extension at 68 ° C for 1 minute. Further, it was kept at 68 ° C. for 2 minutes, and the obtained PCR amplification product was kept at 4 ° C.
  • the DNA fragment thus amplified by PCR was treated with phenol / chloroform, centrifuged, and the supernatant was ethanol precipitated to recover the DNA fragment.
  • the recovered DNA fragment was digested with restriction enzymes SalI and XbaI, and purified and isolated by agarose gel electrophoresis to obtain a gadA promoter DNA fragment.
  • the expression vector pGADP (FIG. 4) was obtained by inserting into the fragment.
  • the expression vector pGADP obtained by the above operation is digested with the restriction enzymes SalI and PstI, purified and isolated by agarose gel electrophoresis, and the DNA fragment of the mutant DOI synthetase gene is ligated and inserted, thereby mutating the DOI synthetase.
  • a molecular population of the gene (mbtrC) was obtained.
  • the molecular population obtained by the above operation was transformed into competent cell cells of E. coli DH5 ⁇ to construct a mutant DOI synthase gene clone library.
  • Example 2 ⁇ Isolation of DOI high production DOI synthase gene mutant clone>
  • a mutant DOI synthetase gene library (pGADP-mbtrC, FIG. 5) is extracted from the mutant DOI synthetase gene clone library obtained by the above operation, and glucose-6-phosphate which is a substrate of DOI synthetase is extracted.
  • Highly accumulating E. coli GI724 ⁇ pgi strain (a strain in which the pgi gene in E. coli GI724 strain is disrupted as described in International Publication No. 2006/109479 and Kakinuma et al., Tetrahedron Letters, 2000, vol. 41, p. 1935) Cells were transformed.
  • the transformed cells were cultured in L agar medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 2% agar, 100 ⁇ g / ml ampicillin), and the grown clones were isolated from DOI synthase gene mutant clones. Selected for separation.
  • L agar medium 1% tryptone, 0.5% yeast extract, 0.5% NaCl, 2% agar, 100 ⁇ g / ml ampicillin
  • the obtained clones to be selected were first selected from RM liquid medium (2% casamino acid, 1% glycerol, 0.6% Na 2 HPO 4 , 0.3% KH 2 PO 4 , 0.05% NaCl. , 0.1% NH 4 Cl, 1 mM MgCl 2 , 100 ⁇ g / ml ampicillin) was inoculated into a deep well plate 96-well round bottom containing 1 ml each, and shaken at 30 ° C. for 24 hours for preculture.
  • RM liquid medium 2% casamino acid, 1% glycerol, 0.6% Na 2 HPO 4 , 0.3% KH 2 PO 4 , 0.05% NaCl. , 0.1% NH 4 Cl, 1 mM MgCl 2 , 100 ⁇ g / ml ampicillin
  • the secondary selection was performed on candidate clones obtained in the primary selection.
  • the candidate clones obtained in the primary selection were inoculated into a test tube containing 3 ml of RM liquid medium, shaken at 30 ° C. for 24 hours, and precultured.
  • 500 ml triangle containing 50 ml each of 2 ⁇ YT liquid medium for secondary selection (1.6% tryptone, 1% yeast extract, 0.5% NaCl, 2% glucose, 100 ⁇ g / ml ampicillin) was used as the preculture.
  • the culture solution at 0, 12, 24, and 36 hours after the start of the main culture was centrifuged, and 10 ⁇ l of the supernatant from which the bacterial cells were removed was subjected to the same operation as the DOI concentration measurement operation in the primary selection, whereby the DOI concentration was selected by HPLC, and a candidate clone having a higher DOI production amount than a clone having a wild type DOI synthase gene having the amino acid sequence of SEQ ID NO: 1 was selected.
  • a tertiary selection was performed on candidate clones obtained in the secondary selection.
  • a plasmid vector was purified and isolated from a clone obtained by secondary selection, and E. coli GI724 ⁇ pgi ⁇ zwf ⁇ pgm strain (International Publication No. 2006/109479 and Kakinuma et al., Tetrahedron) in which glucose-6-phosphate which is a substrate of DOI synthase is highly accumulated.
  • Competent cell cells of pgi gene, zwf gene and pgm gene in E. coli GI724 strain described in Letters, 2000, vol.41, p.1935 were transformed.
  • the transformed cells were cultured in L agar medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 2% agar, 100 ⁇ g / ml ampicillin), and the grown clones were used for the third selection.
  • L agar medium 1% tryptone, 0.5% yeast extract, 0.5% NaCl, 2% agar, 100 ⁇ g / ml ampicillin
  • the third selection first, the obtained clones to be selected were placed in RMM liquid medium (2% casamino acid, 0.5% mannitol, 0.6% Na 2 HPO 4 , 0.3% KH 2 PO 4 , 0.05 % NaCl, 0.1% NH 4 Cl, 1 mM MgCl 2 , 100 ⁇ g / ml ampicillin) was inoculated into a test tube containing 3 ml each, shaken at 30 ° C. for 24 hours, and precultured.
  • RMM liquid medium 2% casamino acid, 0.5% mannitol, 0.6% Na 2
  • the data series represented by ⁇ represents the amount of DOI produced by a clone having the wild-type DOI synthetase gene having the amino acid sequence of SEQ ID NO: 1, and the data series represented by ⁇ represents a clone having the wild-type DOI synthase gene. It is the DOI production amount by the candidate clone with much production amount of DOI.
  • Example 3 ⁇ Analysis of nucleotide sequence of mutant DOI synthase gene> In order to determine the mutation point of the DOI synthetase gene, the base sequence analysis of the DOI synthetase gene of the clone obtained in the third selection was performed.
  • Primers for analysis were primer 7 shown in SEQ ID NO: 8 (5′-ggagccaaccgaagaacc-3 ′), primer 8 shown in SEQ ID NO: 9 (5′-ctagtctagagtcgtttttctgct-3 ′), primer 9 shown in SEQ ID NO: 10 (5′- Using a total of four types of primers, acctgatgcccgaacatg-3 ′) and primer 10 (5′-agatcgaatccgggtccg-3 ′) shown in SEQ ID NO: 11, a PCR reaction was performed using DTCS Quick Start Kit manufactured by Beckman Coulter. The reaction sample was analyzed using a CEQ8000 genetic analyzer manufactured by Beckman Coulter.
  • the 877th T from the start codon of the DOI synthase gene (btrC) was base-substituted. That is, the amino acid substitution of 293th tryptophan from the N-terminal was performed with arginine.
  • a gene (btrC (W293R)) encoding a mutant DOI synthase that improves DOI productivity was obtained as compared with the wild-type DOI synthase having the amino acid sequence of SEQ ID NO: 1.
  • Example 4 Provide and purification of wild type and mutant DOI synthase> A BamHI restriction enzyme site was added upstream of the start codon of the btrC gene using the plasmid vector pLEX-btrC (described in WO 2006/109479) containing the wild-type DOI synthetase of the amino acid sequence of SEQ ID NO: 1 as a template.
  • Primer 11 (5′-cgcggatccatgacgactaaacaaattt-3 ′) shown in SEQ ID NO: 12 corresponding to the nucleotide sequence thus obtained
  • Primer 12 shown in SEQ ID NO: 13 corresponding to the nucleotide sequence added with a HindIII restriction enzyme site upstream of the stop codon of the btrC gene ( 5'-cccaagcttttacagcccttccccgatc-3 ')
  • the nucleotide sequence of the btrC gene was amplified by PCR method, ligated to E. coli recombinant protein high expression vector pQE80L (QIAGEN), transformed into E.
  • the plasmid vector containing the mutant DOI synthetase (btrC (W293R)) obtained above is used as a template and the BamHI restriction enzyme upstream of the start codon of the btrC gene.
  • Primer 11 (5′-cgcggatccatgacgactaaacaaattt-3 ′) shown in SEQ ID NO: 12 corresponding to the nucleotide sequence added with the site, and shown in SEQ ID NO: 13 corresponding to the nucleotide sequence added with a HindIII restriction enzyme site upstream of the stop codon of the btrC gene
  • the nucleotide sequence of btrC (W293R) gene was amplified by PCR using primer 12 (5′-cccaagcttttacagcccttccccgatc-3 ′), ligated to E. coli recombinant protein high expression vector pQE80L (QIAGEN), and transformed into E. coli DH5 ⁇ .
  • plasmid pQE80L-btrC (W293R) was obtained.
  • Recombinant mutant DOI synthase was produced and purified in the same manner.
  • Example 5 ⁇ Measurement of wild-type and mutant DOI synthase activity> The enzyme activities of the wild-type and mutant DOI synthases purified in Example 4 were measured using glucose-6-phosphate and NAD + .
  • the reaction solution is treated with phenol / chloroform, deproteinized, and a method using HPLC used for measuring DOI concentration in the primary selection in Example 2 using 10 ⁇ l of the aqueous layer fraction after centrifugation as a sample.
  • the DOI was quantified and the activity was calculated.
  • the amount of DOI synthesized per 1 minute by 1 mg of DOI synthase was defined as the specific activity.
  • the DOI synthetase activity of the mutant DOI synthetase (W293R) was 1.5 times higher than that of the wild-type DOI synthetase of the amino acid sequence of SEQ ID NO: 1 (FIG. 7).
  • Example 6 In order to obtain a mutant DOI synthetase gene that further improves DOI productivity, a new mutation is obtained by the error-prone PCR method shown in Example 1 using a plasmid containing the mutant DOI synthetase gene (W293R) as a template. Type DOI synthase gene clone library was constructed. Subsequently, the first, second and third selections shown in the isolation of the DOI synthetase gene mutant clone of Example 2 were performed, and the DOI productivity was further improved as compared with the clone containing the mutant DOI synthetase gene (W293R). A clone was obtained (FIG. 8). In FIG.
  • the data series represented by ⁇ represents the DOI production by the E. coli GI724 ⁇ pgi ⁇ zwf ⁇ pgm strain containing pGADP-btrC
  • the data series represented by ⁇ is the DOI production by the E. coli GI724 ⁇ pgi ⁇ zwf ⁇ pgm strain containing pGADP-btrC (W293R).
  • the data series represented by ⁇ , the data series represented by ⁇ , the data series represented by +, and the data series represented by ⁇ are each an E. coli GI724 ⁇ pgi ⁇ zwf ⁇ pgm strain containing different mutant DOI synthase genes. Represents the DOI production by.
  • Table 5 shows the clones containing the wild-type DOI synthase gene, the clones containing the mutant DOI synthase gene having the W293R mutation, and the DOI production (shown in FIG. 8) by each clone shown in Table 4. Indicates a numerical value.
  • the DOI synthetase activity of the mutant DOI synthetase (W293R / N14T) is 1.92 times higher than the activity of the wild-type DOI synthetase of the amino acid sequence of SEQ ID NO: 1 (FIG. 9),
  • the DOI synthetase activity of the mutant DOI synthetase (W293R / Y37F) is 1.57 times higher than the activity of the wild-type DOI synthetase of the amino acid sequence of SEQ ID NO: 1 (FIG.
  • the DOI synthetase activity of the mutant DOI synthetase (W293R / A290T) is 1.45 times higher than the activity of the wild-type DOI synthetase of the amino acid sequence of SEQ ID NO: 1 (FIG. 9)
  • the DOI synthetase activity of the mutant DOI synthetase (W293R / H319R) is 1.83 times higher than the activity of the wild-type DOI synthetase of the amino acid sequence of SEQ ID NO: 1 (FIG. 9)
  • the above four mutant DOI synthetase genes were all more active than the wild-type DOI synthetase having the amino acid sequence of SEQ ID NO: 1.
  • Example 7 ⁇ DOI Fermentation Productivity by Transformant Introducing Mutant DOI Synthase Gene (btrC (W293R / H319R))> An expression vector for expressing a DNA fragment of a mutant DOI synthase gene (btrC (W293R / H319R)) in a host cell (E. coli) was constructed.
  • primer 3 (5′-atggtaccgagctcggatcc-3 ′) shown in SEQ ID NO: 4 and primer 13 (5′-BggHI restriction enzyme site on the 5 ′ side)
  • primer 13 5′-BggHI restriction enzyme site on the 5 ′ side
  • KOD polymerase (TOYOBO) was used for PCR reaction conditions. PCR reaction conditions were maintained at 94 ° C for 2 minutes, followed by 30 cycles of heat denaturation at 94 ° C for 30 seconds, annealing at 50 ° C for 30 seconds, and DNA extension at 68 ° C for 1 minute.
  • the DNA fragment thus amplified by PCR was treated with phenol / chloroform, centrifuged, and the supernatant was ethanol precipitated to recover the DNA fragment.
  • the recovered DNA fragment was digested with restriction enzymes BamHI and PstI, and purified and isolated by agarose gel electrophoresis to obtain an expression vector DNA fragment.
  • a gapA promoter was obtained as a promoter for expressing a DNA fragment of the mutant DOI synthase gene (btrC (W293R / H319R)) in a host cell (E. coli).
  • the gapA promoter was obtained as follows.
  • a primer 14 (5′-cgcggatccgcgggaagagtgaggcgagtc-3 ′) shown in SEQ ID NO: 15 having a BamHI restriction enzyme site on the 5 ′ side and a phosphate group added on the 5 ′ side using a chromosomal DNA of E. coli as a template is shown in SEQ ID NO: 16.
  • PCR amplification was performed using primer 15 (5′-atattccaccacctatttg-3 ′).
  • KOD polymerase TOYOBO
  • PCR reaction conditions were maintained at 94 ° C for 2 minutes, followed by 30 cycles of heat denaturation at 94 ° C for 30 seconds, annealing at 50 ° C for 30 seconds, and DNA extension at 68 ° C for 1 minute. Further, it was kept at 68 ° C. for 2 minutes, and the obtained PCR amplification product was kept at 4 ° C.
  • the DNA fragment thus PCR amplified was treated with phenol / chloroform, centrifuged, and the supernatant was ethanol precipitated to recover the gapA promoter DNA fragment.
  • primer 16 (5′-atgacgactaaacaaatttgttttgcgg-3 ′) shown in SEQ ID NO: 17 with a phosphate group added to the 5 ′ side; PCR amplification was performed using primer 17 (5′-aaaactgcagttacagcccttccggatc-3 ′) shown in SEQ ID NO: 18 having a PstI restriction enzyme site on the 5 ′ side.
  • KOD polymerase TOYOBO
  • PCR reaction conditions were maintained at 94 ° C for 2 minutes, followed by 30 cycles of heat denaturation at 94 ° C for 30 seconds, annealing at 50 ° C for 30 seconds, and DNA extension at 68 ° C for 1 minute. Further, it was kept at 68 ° C. for 2 minutes, and the obtained PCR amplification product was kept at 4 ° C.
  • the PCR-amplified DNA fragment was treated with phenol / chloroform, centrifuged, and the supernatant was ethanol precipitated to recover the DNA fragment of the mutant DOI synthase gene (btrC (W293R / H319R)).
  • PCR reaction conditions were maintained at 94 ° C for 2 minutes, followed by 30 cycles of heat denaturation at 94 ° C for 30 seconds, annealing at 50 ° C for 30 seconds, and DNA extension at 68 ° C for 1 minute. Further, it was kept at 68 ° C. for 2 minutes, and the obtained PCR amplification product was kept at 4 ° C.
  • the DNA fragment thus amplified by PCR was treated with phenol / chloroform, centrifuged, and the supernatant was ethanol precipitated to recover the DNA fragment.
  • the recovered DNA fragment was digested with restriction enzymes BamHI and PstI, and purified and isolated by agarose gel electrophoresis to obtain an expression vector DNA fragment.
  • This DNA fragment was ligated and inserted into the above-described expression vector DNA fragment to obtain plasmid pGAPP-btrC (W293R / H319R) (FIG. 10). Further, pGAPP-btrC in which the wild type DOI synthase gene having the amino acid sequence of SEQ ID NO: 1 was inserted was obtained in the same manner (FIG. 11).
  • Plasmids pGAPP-btrC (W293R / H319R) and pGAPP-btrC were purified and isolated, and E. coli GI724 ⁇ pgi ⁇ zwf ⁇ pgm strain (International Publication No. 2006/109479 and Kakinuma et al.) In which glucose-6-phosphate which is a substrate of DOI synthase is highly accumulated. Tetrahedron Letters, 2000, vol.41, p.1935).
  • the transformed cells are inoculated into a test tube containing 3 ml of 2 ⁇ YT liquid medium (1.6% tryptone, 1% yeast extract, 100 ⁇ g / ml ampicillin), shaken at 30 ° C.
  • the DOI concentration was determined by HPLC.
  • the turbidity of the cells, the glucose concentration in the medium, and the mannitol concentration were also measured.
  • Cellular turbidity is measured at 600 nm using a spectrophotometer, glucose concentration is measured according to glucose CII-Test Wako manufactured by Wako Pure Chemical Industries, and mannitol concentration is measured according to Mannitol Assay Kit manufactured by Magazzyme. did.
  • FIG. 12A shows the time course of the turbidity of the medium
  • FIG. 12B shows the time course of the glucose concentration in the medium
  • FIG. 12C shows the time course of the mannitol concentration in the medium
  • FIG. 12D shows the time course of the DOI production amount.
  • the E. coli GI724 ⁇ pgi ⁇ zwf ⁇ pgm strain containing pGAPP-btrC (W293R / H319R) (data series indicated by ⁇ in FIGS. 12A to 12D) is the E. coli GI724 ⁇ pgi ⁇ zwf ⁇ pgm strain containing pGAPP-btrC (in FIG. 12A to FIG.
  • the DOI production rate was about twice that of the data series.
  • Example 8 ⁇ DOI Fermentation Productivity by Transformant Introducing Mutant DOI Synthase Gene (btrC (H319R))> An expression vector for expressing a DNA fragment of the mutant DOI synthase gene (btrC (H319R)) in a host cell (E. coli) was constructed. Using pGAPP-btrC (FIG.
  • primer 18 (5′-ttccattatttaatccgcgataacaagagg-3 ′) shown in SEQ ID NO: 19 and SEQ ID NO: 20 PCR amplification was performed using primer 19 (5′-cctcttgttatcgcggattaaataatggaa-3 ′).
  • KOD polymerase TOYOBO
  • PCR reaction conditions were maintained at 94 ° C for 2 minutes, followed by 20 cycles of heat denaturation at 98 ° C for 15 seconds, annealing at 55 ° C for 30 seconds, and DNA extension at 68 ° C for 6 minutes. Further, it was kept at 68 ° C. for 3 minutes, and the obtained PCR amplification product was kept at 4 ° C.
  • the DNA fragment thus amplified by PCR was digested with restriction enzyme DpnI and transformed into E. coli DH5 ⁇ to obtain plasmid pGAPP-btrC (H319R) (FIG. 13).
  • Plasmids pGAPP-btrC (H319R) and pGAPP-btrC were purified and isolated, and Escherichia coli GI724 ⁇ pgi ⁇ zwf ⁇ pgm strain (International Publication No. 2006/109479 and Kakinuma et al., Tetrahedron Letters) in which glucose-6-phosphate which is a substrate of DOI synthase is highly accumulated. , 2000, vol.41, p.1935)).
  • the transformed cells are inoculated into a test tube containing 3 ml of 2 ⁇ YT liquid medium (1.6% tryptone, 1% yeast extract, 100 ⁇ g / ml ampicillin), shaken at 30 ° C.
  • the DOI concentration was determined by HPLC.
  • the turbidity of the cells, the glucose concentration in the medium, and the mannitol concentration were also measured. Cellular turbidity is measured at 600 nm using a spectrophotometer, glucose concentration is measured according to glucose CII-Test Wako manufactured by Wako Pure Chemical Industries, and mannitol concentration is measured according to Mannitol Assay Kit manufactured by Magazzyme. did.
  • FIG. 14A shows the time course of turbidity of the medium
  • FIG. 14B shows the time course of the glucose concentration in the medium
  • FIG. 14C shows the time course of the mannitol concentration in the medium
  • FIG. 14D shows the time course of the DOI production amount.
  • the E. coli GI724 ⁇ pgi ⁇ zwf ⁇ pgm strain containing pGAPP-btrC (H319R) (data series represented by ⁇ in FIGS. 14A to 14D) is represented by the E. coli GI724 ⁇ pgi ⁇ zwf ⁇ pgm strain containing pGAPP-btrC ( ⁇ in FIGS. 14A to 14D).
  • the DOI production rate was about 1.2 times that of the data series.
  • Example 9 ⁇ Evaluation of DOI Fermentation Productivity in Jar Fermenter Using Transformants Introduced with Mutant DOI Synthase Genes (btrC (W293R / N14T), (W293R / H319R))>
  • a host and an expression vector different from those in Example 8 were used.
  • E. coli MG1655 ⁇ pgi ⁇ zwf strain in which glucose-6-phosphate which is a DOI synthase substrate is highly accumulated in the host (a strain in which the pgi gene and the zwf gene in E. coli MG1655 strain described in International Publication No. 2010/053052 are disrupted) was used.
  • the expression vector was prepared as follows.
  • Plasmid vector pGAP-btrC-cscA-glf containing DOI synthase gene (btrC) (GATPH promoter described in International Publication No. 2010/053052, btrC which is a DOI synthase gene of Bacillus circulans, E. coli O
  • cscA which is a sucrose hydrolase gene derived from the -157 strain
  • glf which is a glucose transport promoting protein gene derived from Zymomonas mobilis
  • SEQ ID NO: PCR amplification was performed using the primer 20 (5′-catacaggcttttaaataaaatcggg -3 ′) shown in No. 21 and the primer 21 (5′- taaaagcctgtaatgggcggacacgtc-3 ′) shown in SEQ ID NO: 22.
  • PrimeSTAR Max DNA Polymerase (TAKARA) was used for PCR amplification.
  • PCR reaction conditions were 30 cycles of heat denaturation at 98 ° C. for 10 seconds, annealing at 55 ° C. for 15 seconds, and DNA extension reaction at 72 ° C. for 40 seconds as one cycle.
  • the PCR product thus amplified was transformed into E.
  • Primer 22 (5′- tgttttacctttgcattcggcgaacat-3 ′) shown in SEQ ID NO: 23 using the plasmid vector pGAPP-btrC (W293R) -cscA-glf containing the mutant DOI synthase gene (btrC (W293R)) as a template, SEQ ID NO: 24 PCR amplification was performed using the primer 23 (5′-tgcaaaggtaaaacaccggtccgcaaa-3 ′) shown in FIG. PrimeSTAR Max DNA Polymerase (TAKARA) was used for PCR amplification. PCR reaction conditions were 30 cycles of heat denaturation at 98 ° C.
  • primer 24 (5′- ttaatccgcgataacaagaggggctac-3 ′) shown in SEQ ID NO: 25 using the plasmid vector pGAPP-btrC (W293R) -cscA-glf containing the mutant DOI synthase gene (btrC (W293R)) as a template, sequence PCR amplification was performed using primer 25 (5′- ttatcgcggattaaataatggaagat-3 ′) indicated by No. 26. PrimeSTAR Max DNA Polymerase (TAKARA) was used for PCR amplification. PCR reaction conditions were 30 cycles of heat denaturation at 98 ° C.
  • coli MG1655 ⁇ pgi ⁇ zwf strain (described in WO2010 / 053052) in which glucose-6-phosphate which is a substrate of DOI synthase is highly accumulated
  • the three plasmids pGAPP-btrC (W293R) -cscA- were cultured overnight at 37 ° C. on LB agar plates containing 100 ⁇ g / ml ampicillin.
  • lf including pGAPP-btrC (W293R / N14T) -cscA-glf, the pGAPP-btrC (W293R / H319R) -cscA-glf respectively, to obtain three kinds of MG1655 ⁇ pgi ⁇ zwf strains.
  • Various types of MG1655 ⁇ pgi ⁇ zwf strains were inoculated into 0.1 ml of the above-mentioned conical baffle flasks, and agitated and cultured at 120 rpm overnight at 28 ° C.
  • 1 L culture tank (manufactured by ABLE) containing 350 g of medium component 1 (0.2% K 2 HPO 4, 0.2% KH 2 PO 4 , 0.01% FeSO 4 ⁇ 7H 2 O, 0.03% adecanol) Cultivating tank BML-01KP3), 15 g of medium component 2 (10% (NH 4 ) 2 SO 4 , 4.6% NH 4 Cl, 4.6% MgSO 4 .7H 2 O), 50% corn steep 5 g of liquor and 700 ⁇ l of 50% phytic acid were added, 10 g of the preculture was inoculated, and culture was started.
  • medium component 1 (0.2% K 2 HPO 4, 0.2% KH 2 PO 4 , 0.01% FeSO 4 ⁇ 7H 2 O, 0.03% adecanol Cultivating tank BML-01KP3
  • medium component 2 10% (NH 4 ) 2 SO 4 , 4.6% NH 4 Cl, 4.6% MgSO 4 .7H 2 O
  • a reagent sugar solution (21% Glc, 21% Fru, 1% Xyl) was fed at a rate of 0.13 g / min for 30 hours.
  • the reagent sugar solution was prepared by autoclaving and mixing a glucose solution, a fructose solution, and a xylose solution separately.
  • Culturing was performed under atmospheric pressure for 32 hours at an aeration rate of 0.5 L / min, a stirring speed of 800 rpm, a culture temperature of 30 ° C., and a pH of 6.0 (adjusted with a 12.5% ammonia solution). Centrifugation was performed at 0, 8, 24, 27, and 32 hours after the start of the culture, and the supernatant from which the cells had been removed was diluted 100-fold with sterile distilled water and filtered (Mirex-GV, 0.22 ⁇ m, PVDF 4 mm), and the DOI, glucose, fructose, and xylose concentrations were measured using HPLC according to the conditions in Table 7.
  • 18A shows the time course of DOI production ( ⁇ ), glucose concentration ( ⁇ ), fructose concentration ( ⁇ ), and xylose concentration ( ⁇ ) in MG1655 ⁇ pgi ⁇ zwf strain containing pGAPP-btrC (W293R) -cscA-glf.
  • 18B shows the time course of DOI production ( ⁇ ), glucose concentration ( ⁇ ), fructose concentration ( ⁇ ), and xylose concentration ( ⁇ ) in MG1655 ⁇ pgi ⁇ zwf strain containing pGAPP-btrC (W293R / N14T) -cscA-glf.
  • 18C shows the time course of DOI production ( ⁇ ), glucose concentration ( ⁇ ), fructose concentration ( ⁇ ), and xylose concentration ( ⁇ ) in MG1655 ⁇ pgi ⁇ zwf strain containing pGAPP-btrC (W293R / H319R) -cscA-glf. It was.
  • Table 6 shows MG1655 ⁇ pgi32w of strain MG1655 ⁇ pgi32z containing 3 types of plasmids pGAPP-btrC (W293R) -cscA-glf, pGAPP-btrC (W293R / N14T) -cscA-glf, pGAPP-btrC (W293R / H319R) -cscA-glf, respectively.
  • the DOI concentration at the hour is shown.
  • E. coli MG1655 ⁇ pgi ⁇ zwf strain containing pGAPP-btrC (W293R) -cscA-glf E.
  • the DOI concentration at 32 hours was 56.6 g / L for MG1655 ⁇ pgi ⁇ zwf strain containing pGAPP-btrC (W293R) -cscA-glf, and 30.6 g for MG1655 ⁇ pgi ⁇ zwL strain containing pGAPP-btrC (W293R / N14T) -cscA-glf.
  • MG1655 ⁇ pgi ⁇ zwf strain containing pGAPP-btrC (W293R / H319R) -cscA-glf was 70.5 g / L.
  • the MG1655 ⁇ pgi ⁇ zwf strain containing pGAPP-btrC (W293R / H319R) -cscA-glf showed about 1.2 times the DOI productivity of the MG1655 ⁇ pgi ⁇ zwf strain containing pGAPP-btrC (W293R) -cscA-glf.

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Abstract

L'invention concerne un polypeptide qui présente une mutation d'acide aminé spécifique au niveau d'au moins l'un des résidus d'acides aminés situés en position 14, 37, 290, 293 et 319, en partant de l'extrémité N-terminale, de la séquence d'acides aminés représentée par SEQ ID NO : 1 dans la séquence d'acides aminés représentée par SEQ ID NO : 1 ou une séquence analogue à celle-ci ; un polynucléotide comportant une séquence de bases codant pour la séquence d'acides aminés du polypeptide mentionné ci-dessus ; une cassette d'expression ; un vecteur ; un transformant ; un procédé de production dudit polypeptide ; et un procédé de production de 2-désoxy-scyllo-inosose.
PCT/JP2018/010349 2017-03-27 2018-03-15 2-désoxy-scyllo-inosose synthase de type mutant Ceased WO2018180568A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2022513056A (ja) * 2018-11-15 2022-02-07 バイオアプリケーションズ インコーポレイテッド 植物体でウイルス様粒子を発現する組み換えベクター及びこれを利用したサーコウイルス様粒子を含むワクチン組成物の製造方法
JP7212968B2 (ja) 2018-11-15 2023-01-26 バイオアプリケーションズ インコーポレイテッド 植物体でウイルス様粒子を発現する組み換えベクター及びこれを利用したサーコウイルス様粒子を含むワクチン組成物の製造方法
US12156907B2 (en) 2018-11-15 2024-12-03 Bioapplications Inc. Recombinant vector for expressing virus-like particles in plant and method for preparation of vaccine composition containing circovirus-like particles by using same

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